Protein farnesyltransferase (FTase) is a key enzyme that is responsible for the post-translational modification of a number of proteins involved in cell growth. These proteins include ras proteins, nuclear lamins and the yeast a-mating factor, all of which end with a unique C-terminal sequence, (SEQ. ID. NO. 1) CAAX (where A is an aliphatic amino acid and X is the C-terminal amino acid) termed the `CAAX box`. FTase catalyses the addition of a farnesyl group to the cysteine.
Ras proteins have been implicated in oncogenesis. Ras proteins are farnesyl acceptors. In the case of ras proteins, farnesylation contributes to an increase in their hydrophobicity, thus facilitating their membrane localization. Because a proper membrane localization is required for the function of ras proteins, inhibiting their membrane localization may block the action of oncogenic forms of the ras protein.
Several approaches to block ras function have been proposed. These include reversal of impaired GTPase activity of the oncogenic ras molecule and inhibition of ras interaction with its target or effector molecule. The most promising may be to interfere at the level of ras membrane localization by using specific drugs that inhibit ras FTase. Inhibitors of FTase so far reported include prenyl substrate analogues or synthetic peptides that correspond to the ras CAAX motif. However, intracellular delivery of these inhibitors may present a problem. For prenyl substrate analogues, the diphosphate prevents cell penetration. As for peptides, cellular uptake may also be inefficient and degradation in intestinal cells may be rapid. Non-peptide and non-prenyl diphosphate type inhibitors are suitable for development as potent orally active long-duration therapeutic agents.
Inhibition of ras farnesylation in vivo was demonstrated with compactin, but, this drug inhibits HMG-CoA reductase and acts as a general inhibitor of the isoprenoid pathway, and is unlikely to be useful for specifically antagonizing ras function. On the other hand, an inhibitor of ras farnesyltransferase, the key enzyme that catalyzes the farnesylation, would not perturb other elements of the mevalonate pathway, and therefore, would be expected to be a more effective antagonist of ras function. Searches for such inhibitors requires detection assays and methods of confirming the specificity and kinetics of the inhibitors.
FTase from rat brain has been characterized. This enzyme has strong affinity to the CAAX sequence. It binds to a column containing peptides having the CAAX sequence and is inhibited by tetrapeptides containing the CAAX sequence. The tetrapeptide inhibition was useful in the determination of sequence requirements for the CAAX box. The mammalian enzyme is a heterodimer consisting of two similar sized subunits termed .alpha. and .beta.. The .beta. subunit can be cross-linked to ras proteins, suggesting that the site of CAAX recognition is within the .beta.-subunit. Purification of FTase from this source requires peptide affinity chromatography as a final step. This step is difficult to reproduce. Large-scale production of purified mammalian enzyme is not available, in particular, by recombinant technology.
An FTase similar to the mammalian enzyme was detected in crude extracts of yeast cells. Two genes, designated DPR1 or RAM1 (referred to herein as DPR1), and RAM2, respectively, are required for the yeast FTase activity. A possible explanation is that the enzyme consists of two subunits encoded by these genes. This hypothesis is strengthened by the detection of a significant (approximately 30%) identity between the expression product of DPR1 and the .beta.-subunit of the mammalian FTase as well as between the expression product of RAM2 and the .alpha.-subunit of the mammalian FTase. Levels of expression, however, are too low for adequate processing, for example, purification. FTase activity of crude extracts was also detected by transfer activity of farnesyl onto a substrate after simultaneous expression of DPR1 and RAM2 genes in Escherichia coli, but the enzyme was neither overproduced nor purified.
In the present invention a surrogate for mammalian FTase was sought. Recombinant technology was used to overproduce FTase. Yeast was selected as a host cell to attempt to enhance production of FTase so that sufficient protein was available for purification. Multiple copies of genes for both subunits of protein FTase were introduced into the host cell to overproduce the enzyme. Unexpected synergism resulted, making it possible to achieve the purification goal. FTase was found to be an acceptable surrogate for mammalian FTase in tests for inhibitors. Either mammalian or yeast gene sequences may be used for expressing the enzyme. A purification involving one column is all that is subsequently required to purify the enzyme to near homogeneity. The purified enzyme is useful for confirming the specifity and kinetics of FTase inhibitors.